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BACKGROUND/AIM: In vivo imaging with luciferase-luciferin has been limited by the inability to visualize the low emitted light, with the signal quantified only by photon counting using a cumbersome highly-cooled CCD camera in a dark room. In the present study, we demonstrate direct visualization of the luciferase-luciferin signal from an orthotopic lung cancer in a nude-mouse xenograft model with a sensitive low-light camera and optics. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells expressing luciferase (LL/2-Luc2) were injected transcutaneously into the lung of a nude mouse. One week later after cell injection, luciferase imaging for emission at 560 nm was performed using the UVP Biospectrum Advanced system after i.v. injection of D-luciferin potassium salt. The intensity of the visualized light was measured and quantified with the instrument. RESULTS: A week following the implantation of LL/2-Luc2 cells in nude mice, the luciferase-luciferin signal from LL/2-Luc2 tumors in the lung was sufficiently visible through the skin to produce true images. At fifteen minutes, the intensity peaked and then progressively dropped due to clearance of luciferin from the tumor. CONCLUSION: Using the UVP Biospectrum Advanced system we demonstrated non-invasive visualization of true images from luciferase-luciferin signals from an orthotopic lung-cancer mouse model. The luciferase-luciferin emitted light was directly visible through the skin which is a major improvement over previous photon counting to detect the luciferase-luciferin signal.
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Luciferasas , Mediciones Luminiscentes , Neoplasias Pulmonares , Ratones Desnudos , Animales , Ratones , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/diagnóstico por imagen , Luciferasas/metabolismo , Luciferasas/genética , Línea Celular Tumoral , Mediciones Luminiscentes/métodos , Modelos Animales de Enfermedad , Humanos , Xenoinjertos , Benzotiazoles , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/AIM: Exosome exchange between cancer cells or between cancer and stromal cells is involved in cancer metastasis. We have previously developed in vivo color-coded labeling of cancer cells and stromal cells with spectrally-distinct fluorescent genetic reporters to demonstrate the role of exosomes in metastasis. In the present study, we studied exosome transfer between different pancreatic-cancer cell lines in vivo and in vitro and its potential role in metastasis. MATERIALS AND METHODS: Human pancreatic-cancer cell lines AsPC-1 and MiaPaCa-2 were used in the present study. AsPC-1 cells contain a genetic exosome reporter gene labeled with green fluorescent protein (pCT-CD63-GFP) and MiaPaCa-2 cells express red fluorescent protein (RFP). Both cell lines were co-injected into the spleen of nude mice (n=5) to further study the role of exosome exchange in metastasis. Three weeks later mice were sacrificed and tumors at the primary and metastatic sites were cultured and observed by confocal fluorescence microscopy for exosome transfer. RESULTS: The primary tumor formed in the spleen and metastasized to the liver, as observed macroscopically. Cells were cultured from the spleen, liver, lung, bone marrow and ascites. Transfer of exosomes from AsPC-1 to MiaPaCa-2 was demonstrated in the cultured cells by confocal fluorescence microscopy. Moreover, cell fusion was also observed along with exosome transfer. Exosome transfer did not occur during in vitro co-culture between the two pancreatic-cancer cell lines, suggesting a role of the tumor microenvironment (TME) in exosome transfer. CONCLUSION: The transfer of exosomes between different pancreatic-cancer cell lines was observed during primary-tumor and metastatic growth in nude mice. This cell-cell communication might be a trigger of cell fusion and promotion of cancer metastasis. Exosome transfer between the two pancreatic-cancer cell lines appears to be facilitated by the TME, as it did not occur during in vitro co-culture.
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Técnicas de Cocultivo , Exosomas , Ratones Desnudos , Neoplasias Pancreáticas , Exosomas/metabolismo , Animales , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Humanos , Línea Celular Tumoral , Ratones , Metástasis de la Neoplasia , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genética , Proteína Fluorescente Roja , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genéticaRESUMEN
Determining the safety of a newly developed experimental product is a crucial condition for its medical use, especially for clinical trials. In this regard, four hydrogel-type formulations were manufactured, all of which were based on carbomer (Blank-CP940) and encapsulated with caffeine (CAF-CP940), phosphorus derivatives (phenyl phosphinic (CAF-S1-CP940) and 2-carboxyethyl phenyl phosphinic acids (CAF-S2-CP940)). The main aim of this research was to provide a comprehensive outline of the biosafety profile of the above-mentioned hydrogels. The complex in vitro screening (cell viability, cytotoxicity, morphological changes in response to exposure, and changes in nuclei morphology) on two types of healthy skin cell lines (HaCaT-human keratinocytes and JB6 Cl 41-5a-murine epidermal cells) exhibited a good biosafety profile when both cell lines were treated for 24 h with 150 µg/mL of each hydrogel. A comprehensive analysis of the hydrogel's impact on the genetic profile of HaCaT cells sustains the in vitro experiments. The biosafety profile was completed with the in vivo and in ovo assays. The outcome revealed that the developed hydrogels exerted good biocompatibility after topical application on BALB/c nude mice's skin. It also revealed a lack of toxicity after exposure to the hen's chicken embryo. Further investigations are needed, regarding the in vitro and in vivo therapeutic efficacy and safety for long-term use and potential clinical translatability.
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Antitumor drugs used today have shown significant efficacy and are derived from natural products such as plants. Iso-mukaadial acetate (IMA) has previously been shown to possess anticancer properties by inducing apoptosis. The purpose of this study was to investigate the therapeutic effect of IMA in the breast cancer xenograft mice model. Female athymic nude mice were used and inoculated with breast cancer cells subcutaneously. Untreated group one served as a negative control and positive control group two (cisplatin) was administered intravenously. IMA was administered orally to group three (100 mg/kg) and group four (300 mg/kg). Blood was collected (70 µL) from the tail vein on day zero, day one and day three. Tumor regression was measured every second day and body mass was recorded each day. Estimation of serum parameters for renal indices was examined using a creatinine assay. Histopathological analysis was conducted to evaluate morphological changes of liver, kidney, and spleen tissues before and after compound administration under a fluorescence light microscope. Histopathological analysis of tumors was conducted before and after compound administration. Apoptotic analysis using the TUNEL system was conducted on liver, kidney, and spleen tissues. Tumor shrinkage and reduction in body mass were observed after treatment with IMA. Serum creatinine was slightly elevated after treatment with IMA at a dosage of 100 and 300 mg/kg. Histopathological results of the liver exhibited no changes before and after IMA while the kidney and spleen tissues showed changes in the cellular structure. IMA showed no cytotoxic effect on the tumor cells, and cell proliferation was observed. Apoptotic assay stain with TUNEL showed apoptotic cells in spleen tissue and kidney but no apoptotic cells were observed in liver tissue section treated with IMA. IMA showed clinical toxic signs that resulted in the suffering and death of the mice immediately after IMA administration. Histopathology of tumor cells showed that IMA did not inhibit cell proliferation and no cellular damage was observed. Therefore, based on the results obtained, we cannot make any definitive conclusion on the complete effect of IMA in vivo. IMA is toxic, poorly soluble, and not safe to use in animal studies. The objective of the study was not achieved, and the hypothesis was rejected.
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Apoptosis , Neoplasias de la Mama , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Humanos , Femenino , Ratones , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Apoptosis/efectos de los fármacos , Células MCF-7 , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacosRESUMEN
BACKGROUND/AIM: Methotrexate (MTX) resistance in osteosarcoma leads to a very poor prognosis. In the present study, in order to further understand the basis and ramifications of MTX resistance in osteosarcoma, we selected an osteosarcoma cell line that has a 5,500-fold-increased MTX IC50 Materials and Methods: The super MTX-resistant 143B osteosarcoma cells (143B-MTXSR) were selected from MTX-sensitive parental human 143B osteosarcoma cells (143B-P) by continuous culture with step-wise increased amounts of MTX. To compare the malignancy of 143B-MTXSR and 143B-P, colony-formation capacity was compared with clonogenic assays on plastic and in soft agar. In addition, tumor growth was compared with orthotopic xenograft mouse models of osteosarcoma. Expression of dihydrofolate reductase (DHFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and myelocytomatosis oncogene (MYC) was examined with western immunoblotting and compared in 143B-MTXSR and 143B-P cells. RESULTS: 143B-MTXSR had a 5,500-fold increase in the MTX IC50 compared to the parental 143B-P cells. Expression of DHFR was increased 10-fold in 143B-MTXSR compared to 143B-P (p<0.01). 143B-MTXSR cells had reduced colony-formation capacity on plastic (p=0.032) and in soft agar (p<0.01) compared to 143B-P and reduced tumor growth in orthotopic xenograft mouse models (p<0.001). These results demonstrate that 143B-MTXSR had reduced malignancy. 143B-MTXSR also showed an increased expression of PI3K (p<0.01), phosphorylated (activated) AKT (p=0.031), phosphorylated mTOR (p=0.043), and c-MYC (p=0.024) compared to 143B-P. CONCLUSION: The present study demonstrates that the increased expression of DHFR, PI3K/AKT/mTOR and c-MYC appears to be linked to super MTX resistance and, paradoxically, to reduced malignancy. The present results suggest that DHFR may be a powerful tumor suppressor when highly amplified.
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Resistencia a Antineoplásicos , Metotrexato , Osteosarcoma , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-myc , Serina-Treonina Quinasas TOR , Tetrahidrofolato Deshidrogenasa , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Osteosarcoma/metabolismo , Osteosarcoma/genética , Metotrexato/farmacología , Humanos , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/genética , Amplificación de Genes , Transducción de Señal/efectos de los fármacos , Ratones Desnudos , Antimetabolitos Antineoplásicos/farmacologíaRESUMEN
Personalized medicine is a new approach to modern oncology. Here, to facilitate the application of extracellular vesicles (EVs) derived from lung cancer cells as potent advanced therapy medicinal products in lung cancer, the EV membrane was functionalized with a specific ligand for targeting purposes. In this role, the most effective heptapeptide in binding to lung cancer cells (PTHTRWA) was used. The functionalization process of EV surface was performed through the C- or N-terminal end of the heptapeptide. To prove the activity of the EVs functionalized with PTHTRWA, both a model of lipid membrane mimicking normal and cancerous cell membranes as well as human adenocarcinomic alveolar basal epithelial cells (A549) and human normal bronchial epithelial cells (BEAS-2B) have been exposed to these bioconstructs. Magnetic resonance imaging (MRI) showed that the as-bioengineered PTHTRWA-EVs loaded with superparamagnetic iron oxide nanoparticle (SPIO) cargos reach the growing tumor when dosed intravenously in NUDE Balb/c mice bearing A549 cancer. Molecular dynamics (MD) in silico studies elucidated a high affinity of the synthesized peptide to the α5ß1 integrin. Preclinical safety assays did not evidence any cytotoxic or genotoxic effects of the PTHTRWA-bioengineered EVs.
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Vesículas Extracelulares , Neoplasias Pulmonares , Ratones Endogámicos BALB C , Ratones Desnudos , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Animales , Ratones , Células A549 , Nanopartículas Magnéticas de Óxido de Hierro/químicaAsunto(s)
Adhesión Celular , Neoplasias del Colon , Neoplasias Hepáticas , Proteínas Asociadas a Pancreatitis , Humanos , Neoplasias del Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Línea Celular Tumoral , Proteínas Asociadas a Pancreatitis/metabolismo , Proteínas Asociadas a Pancreatitis/genética , Animales , Regulación Neoplásica de la Expresión Génica , RatonesRESUMEN
Cancer remains a worldwide cause of morbidity and mortality. Investigational research efforts have included the administration of tumor-derived extracts to healthy animals. Having previously demonstrated that the administration of non-transmissible, human cancer-derived homogenates induced malignant tumors in mice, here, we examined the consequences of administering 50 or 100 µg of protein of crude homogenates from mammary carcinoma, pancreatic adenocarcinoma, and melanoma samples in 6 inoculations per week during 2 months. The concurrent control mice received homogenates of healthy donor-skin cosmetic surgery fragments. Mammary carcinoma homogenate administration did not provoke the deterioration or mortality of the animals. Multiple foci of lung adenocarcinomas with a broad expression of malignity histomarkers coexisting with small cell-like carcinomas were found. Disseminated cells, positive to classic epithelial markers, were detected in lymphoid nodes. The administration of pancreatic tumor and melanoma homogenates progressively deteriorated animal health. Pancreatic tumor induced poorly differentiated lung adenocarcinomas and pancreatic islet hyperplasia. Melanoma affected lungs with solid pseudopapillary adenocarcinomas. Giant atypical hepatocytes were also observed. The kidney exhibited dispersed foci of neoplastic cells within a desmoplastic matrix. Nuclear overlapping with hyperchromatic nuclei, mitotic figures, and prominent nuclear atypia was identified in epidermal cells. None of these changes were ever detected in the control mice. Furthermore, the incubation of zebrafish embryos with breast tumor homogenates induced the expression of c-Myc and HER-2 as tumor markers, contrasting to embryos exposed to healthy tissue-derived material. This study confirms and extends our hypothesis that tumor homogenates contain and may act as vectors for "malignancy drivers," which ultimately implement a carcinogenesis process in otherwise healthy mice.
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BACKGROUND/AIM: Colorectal cancer (CRC) is the third-leading cause of death in the world. Although the prognosis has improved due to improvement of chemotherapy, metastatic CRC is still a recalcitrant disease, with a 5-year survival of only 13%. Irinotecan (IRN) is used as first-line chemotherapy for patients with unresectable CRC. However, there are severe side effects, such as neutropenia and diarrhea, which are dose-limiting. We have previously shown that methionine restriction (MR), effected by recombinant methioninase (rMETase), lowered the effective dose of IRN of colon-cancer cells in vitro. The aim of the present study was to evaluate the efficacy of the combination of low-dose IRN and MR on colon-cancer in nude mice. MATERIALS AND METHODS: HCT-116 colon-cancer cells were cultured and subcutaneously injected into the flank of nude mice. After the tumor size reached approximately 100 mm3, 18 mice were randomized into three groups; Group 1: untreated control on a normal diet; Group 2: high-dose IRN on a normal diet (2 mg/kg, i.p.); Group 3: low-dose IRN (1 mg/kg i.p.) on MR effected by a methionine-depleted diet. RESULTS: There was no significant difference between the control mice and the mice treated with high-dose IRN, without MR. However, low-dose IRN combined with MR was significantly more effective than the control and arrested colon-cancer growth (p=0.03). Body weight loss was reversible in the mice treated by low-dose IRN combined with MR. CONCLUSION: The combination of low-dose IRN and MR acted synergistically in arresting HCT-116 colon-cancer grown in nude mice. The present study indicates the MR has the potential to reduce the effective dose of IRN in the clinic.
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Liasas de Carbono-Azufre , Neoplasias del Colon , Irinotecán , Metionina , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Irinotecán/administración & dosificación , Irinotecán/farmacología , Metionina/administración & dosificación , Humanos , Ratones , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Camptotecina/análogos & derivados , Camptotecina/farmacología , Camptotecina/administración & dosificación , Camptotecina/uso terapéutico , Modelos Animales de Enfermedad , Células HCT116 , Línea Celular Tumoral , Carga Tumoral/efectos de los fármacosRESUMEN
Negative pressure therapy (NPT) has been shown to facilitate wound healing and promote hair growth in a porcine model. However, there is a paucity of research on the impact of negative pressure on hair growth in murine models. Despite the ability of nude mice to develop hair follicles, the hair they produce is often flawed towing to genetically induced keratin disorders, rendering them a pertinent animal model for assessing hair regeneration. Therefore, this study aims to investigate the effects of negative pressure on hair follicle growth in a nude mouse model. To achieve this, a customized external tissue expansion device was developed to apply negative pressure to the dorsum of nude mice. The mice were subjected to several treatment courses consisting of 15 and 30 min of continuous negative pressure at 10 mmHg, which were repeated 5 and 10 times every other day until sacrifice. Dorsal skin samples were subsequently extracted from the suction and nonsuction areas. The sections were stained with various antibodies to assess the expression of SOX-9, LHX-2, Keratin-15, ß-catenin, CD31, and vascular endothelial growth factor-A, and a TUNEL assay was used to analyze cell apoptosis. The results showed that the number of hair follicles and angiogenesis were significantly higher in the suction area than in the nonsuction area in all groups. Moreover, mice that received NPT for 15 min for 10 times had a higher hair follicle density than the other three groups. Immunofluorescence staining for LHX-2 and Keratin 15 further validated the results of these findings. In conclusion, this study demonstrated that negative pressure effectively promotes hair follicle growth and angiogenesis in nude mice through SOX-9- and LHX-2-mediated follicular regeneration and ß-catenin-mediated hair follicle morphogenesis.
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Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti-tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti-proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.
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Cannabinoides , Cannabis , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Ratones , Humanos , Cannabinoides/farmacología , Cannabinoides/uso terapéutico , Ratones Desnudos , Xenoinjertos , Proteína X Asociada a bcl-2 , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
BACKGROUND/AIM: Fat grafting has been widely used for soft-tissue augmentation. External volume expansion (EVE) is a favorable tool for improvement in the rate of fat graft retention. However, few studies have focused on the most appropriate time for its implementation. In this study, BALB/c nude mice were used to investigate the effective time for the implementation of external volume expansion to improve the rate of fat retention. MATERIALS AND METHODS: Sixteen mice were divided into four groups, and EVE was performed at different time points before or both before and after fat grafting. Fat tissue from a human donor was injected into the mice following EVE. Visual assessment, micro-computed tomography analysis, and histopathological evaluation were used to assess fat retention. RESULTS: After 10 weeks, the group that underwent EVE 5 days before fat grafting demonstrated a significantly higher preserved fat volume, as determined by micro-computed tomography (p<0.05). Moreover, the group that received additional EVE after fat grafting exhibited a higher retention rate compared to the groups receiving EVE only before grafting (p<0.05). Histopathological analysis indicated that swelling, edema, and inflammation were more pronounced in the group with EVE immediately before grafting, while angiogenesis and lipogenesis were more active in the group with additional EVE after grafting. CONCLUSION: EVE is a safe and effective approach for improving the rate of fat graft retentions. Furthermore, the timing of external tissue expansion plays a crucial role in fat retention. Based on our animal study, performing EVE immediately before and after fat grafting may be an effective strategy for enhancing the rate of fat graft retentions.
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Tejido Adiposo , Inflamación , Animales , Ratones , Humanos , Ratones Desnudos , Microtomografía por Rayos X , Tejido Adiposo/trasplante , Supervivencia de InjertoRESUMEN
Breast cancer is a dreaded disease affecting women the most in cancer-related deaths over other cancers. However, early diagnosis of the disease can help increase survival rates. The existing breast cancer diagnosis tools do not support the early diagnosis of the disease. Therefore, there is a great need to develop early diagnostic tools for this cancer. Photoacoustic spectroscopy (PAS), being very sensitive to biochemical changes, can be relied upon for its application in detecting breast tumors in vivo. With this motivation, in the current study, an aseptic chamber integrated photoacoustic (PA) probe was designed and developed to monitor breast tumor progression in vivo, established in nude mice. The device served the dual purpose of transporting tumor-bearing animals to the laboratory from the animal house and performing PA experiments in the same chamber, maintaining sterility. In the current study, breast tumor was induced in the nude mice by MCF-7 cells injection and the corresponding PA spectra at different time points (day 0, 5, 10, 15, and 20) of tumor progression in vivo in the same animals. The recorded photoacoustic spectra were subsequently preprocessed, wavelet-transformed, and subjected to filter-based feature selection algorithm. The selected top 20 features, by minimum redundancy maximum relevance (mRMR) algorithm, were then used to build an input feature matrix for machine learning (ML)-based classification of the data. The performance of classification models demonstrated 100% specificity, whereas the sensitivity of 95, 100, 92.5, and 85% for the time points, day 5, 10, 15, and 20, respectively. These results suggest the potential of PA signal-based classification of breast tumor progression in a preclinical model. The PA signal contains information on the biochemical changes associated with disease progression, emphasizing its translational strength toward early disease diagnosis.
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Neoplasias de la Mama , Animales , Ratones , Humanos , Femenino , Ratones Desnudos , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Algoritmos , Aprendizaje Automático , Análisis EspectralRESUMEN
Background: Esophageal squamous cell carcinoma (ESCC) is a common pathological esophageal cancer with poor prognosis. Vitamin D deficiency reportedly occurs in ESCC patients, and this is related to single nucleotide polymorphism of vitamin D receptor (VDR). Objective: We investigated the effect of VDR on ESCC proliferation, invasion, and metastasis and its potential mechanism. Methods: ESCC and normal tissues were collected from 20 ESCC patients. The ESCC tissue microarray contained 116 pairs of ESCC and normal tissues and 73 single ESCC tissues. VDR expression and its clinicopathological role were determined by real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry staining. sh-VDR and VDR overexpression were used to validate the effect of VDR on ESCC cell phenotype, and tandem mass tag-based quantitative proteomics and bioinformatics methods identified differential VDR-related proteins. The downstream pathway and regulatory effect were analyzed using ingenuity pathway analysis (IPA). Differentially expressed proteins were verified through parallel reaction monitoring and Western blot. In vivo imaging visualized subcutaneous tumor growth following tail vein injection of VDR-deficient ESCC cells. Results: High VDR expression was observed in ESCC tissues and cells. Gender, T stage, and TNM stage were related to VDR expression, which was the independent prognostic factor related to ESCC. VDR downregulation repressed ESCC cell proliferation, invasion, and migration in vitro and subcutaneous tumor growth and lung metastases in vivo. The cell phenotype changes were reversed upon VDR upregulation, and differential proteins were mainly enriched in the p53 signaling pathway. TP53 cooperated with ABCG2, APOE, FTH1, GCLM, GPX1, HMOX1, JUN, PRDX5, and SOD2 and may activate apoptosis and inhibit oxidative stress, cell metastasis, and proliferation. TP53 was upregulated after VDR knockdown, and TP53 downregulation reversed VDR knockdown-induced cell phenotype changes. Conclusions: VDR may inhibit p53 signaling pathway activation and induce ESCC proliferation, invasion, and metastasis by activating oxidative stress.
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Background/Aim: Pancreatic cancer is a recalcitrant disease with 5-year survival of only 12%. Improved mouse models of pancreatic cancer are critical for discovery of effective therapeutics. Materials and Methods: Orthotopic mouse nude-mouse models of pancreatic cancer were established with the human pancreatic-cancer cell line Panc-1 expressing green fluorescent protein (GFP) by transplanting tumor fragments into the pancreas, using the procedure of surgical orthotopic implantation (SOI). Four weeks after establishment of the orthotopic models, the mice were imaged with the Analytik Jena UVP Biospectrum Advanced with a very-narrow-band-width excitation at 487 nm and peak emission at 513 nm. Results: Non-invasive fluorescence imaging of the mice implanted with Panc-1-GFP showed a very bright tumor in the area of the pancreas and peritoneal cavity. The skin background autofluorescence was absent. When a laparotomy was performed on the mouse for open imaging, the tumor on the pancreas was clearly imaged. There was very clear concordance of the non-invasive image and the image obtained during laparotomy. Conclusion: A precise orthotopic mouse model of pancreatic cancer was developed in which there was high concordance between non-invasive and invasive fluorescence imaging due to the ultra-bright signal and ultra-low background using very-narrow-band-width laser fluorescence excitation. This model can be used for high-throughput in vivo screening for improved therapeutics for pancreatic cancer.
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Cell-based liver therapies utilizing functionally stabilized engineered hepatic tissue hold promise in improving host liver functions and are emerging as a potential alternative to whole-organ transplantation. Owing to the ability to accommodate a large ex vivo engineered hepatocyte mass and dense vascularization, the mesenteric parametrial fat pad in female nude mice forms an ideal anatomic microenvironment for ectopic hepatocyte transplantation. However, the lack of any reported protocol detailing the presurgical preparation and construction of the engineered hepatic hydrogel, fat pad surgery, and postsurgical care and bioluminescence imaging to confirm in vivo hepatocyte implantation makes it challenging to reliably perform and test engraftment and integration with the host. In this report, we provide a step-by-step protocol for in vivo hepatocyte implantation, including preparation of hepatic tissue for implantation, the surgery process, and bioluminescence imaging to assess survival of functional hepatocytes. This will be a valuable protocol for researchers in the fields of tissue engineering, transplantation, and regenerative medicine. Key features ⢠Primary human hepatocytes transduced ex vivo with a lentiviral vector carrying firefly luciferase are surgically implanted onto the fat pad. ⢠Bioluminescence helps monitor survival of transplanted hepatic tissue over time. ⢠Applicable for assessment of graft survival, graft-host integration, and liver regeneration.
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Glioma is a highly recalcitrant disease with a 5-year survival of 6.8 %. Temozolomide (TMZ), first-line therapy for glioma, is more effective in O6-methylguanine-DNA methyltransferase (MGMT)-negative gliomas than in MGMT-positive gliomas as MGMT confers resistance to TMZ. Methionine restriction is effective for many cancers in mouse models including glioma. The concern is that methionine restriction could induce MGMT by decreasing DNA methylation and confer resistance to TMZ. In the present study, we investigated the efficacy of combining methionine restriction with TMZ for the treatment of MGMT-negative glioma, and whether methionine restriction induced MGMT. Human MGMT-negative U87 glioma cells were used to determine the efficacy of TMZ combined with methionine restriction. Recombinant methioninase (rMETase) inhibited U87 glioma growth without induction of MGMT in vitro. The combination of rMETase and TMZ inhibited U87 cell proliferation more than either agent alone in vitro. In the orthotopic nude-mouse model, the combination of TMZ and a methionine-deficient diet was much more effective than TMZ alone: two mice out of five were cured of glioma by the combination. No mice died during the treatment period. Methionine restriction enhanced the efficacy of TMZ in MGMT-negative glioma without inducing MGMT, demonstrating potential clinical promise for improved outcome of a currently incurable disease.
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Neoplasias Encefálicas , Glioma , Temozolomida , Animales , Humanos , Ratones , Antineoplásicos Alquilantes/uso terapéutico , Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Metilasas de Modificación del ADN/farmacología , Metilasas de Modificación del ADN/uso terapéutico , Enzimas Reparadoras del ADN/genética , Resistencia a Antineoplásicos , Glioma/tratamiento farmacológico , Glioma/genética , Metionina/farmacología , Ratones Desnudos , O(6)-Metilguanina-ADN Metiltransferasa , Racemetionina/farmacología , Temozolomida/uso terapéutico , Temozolomida/farmacología , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND/AIM: Breast-cancer metastasis to the brain is an intractable disease. To discover improved therapy for this disease, we developed a precise non-invasively-imageable orthotopic nude-mouse model, using very-narrow-band-width laser fluorescence excitation. MATERIALS AND METHODS: Female nu/nu nude mice, aged 4-8 weeks, were inoculated through the midline of the skull with triple-negative human MDA-MB-231 breast cancer cells (5×105) expressing red fluorescent protein (RFP). The mice were imaged with the Analytik Jena UVP Biospectrum Advanced at 520 nm excitation with peak emission at 605 nm. RESULTS: Three weeks after injection of MDA-MB-231-RFP cells in the brain, non-invasive fluorescence images of the breast tumor growing on the brain were obtained. The images of the tumor were very bright, with well-defined margins with no detectable skin autofluorescence background. Images obtained at various angles showed that the extent of the tumor margins could be precisely measured. A skin flap over the skull confirmed that the tumor was growing on the surface of the brain which is a frequent occurrence in breast cancer. CONCLUSION: A precise orthotopic model of RFP-expressing breast-cancer metastasis to the brain was developed that could be non-invasively imaged with very-narrow-band-width laser excitation, resulting in an ultra-bright, ultra-low-background signal. The model will be useful in discovering improved therapeutics for this recalcitrant disease.
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Neoplasias de la Mama , Melanoma , Neoplasias Primarias Secundarias , Neoplasias Cutáneas , Ratones , Femenino , Humanos , Animales , Proteína Fluorescente Roja , Neoplasias de la Mama/diagnóstico por imagen , Ratones Desnudos , Modelos Animales de Enfermedad , Imagen Óptica , Encéfalo/diagnóstico por imagen , Proteínas Fluorescentes Verdes , Línea Celular TumoralRESUMEN
Objective To investigate the effect and mechanism of miR-20a-5p on human nephroblastoma cell line WiT49 transplanted tumor in nude mice.Methods The gene expression chip was downloaded from GEO database,and the differential gene miR-20a-5p was obtained by GEO2R.The NF-κB gene was positively correlated with the expression of miR-20a-5p through cBioPortal database.The target gene of miR-20a-5p was predicted to be NFKBIB of the NF-κB transcription factor suppressor protein family by targetscan database,and was verified by dual luciferase assay.Nephroblastoma cell line WiT49 was cultured in vitro and transfected into WiT49 cells with lentiviral vectors constructed with miR-20a-5p mimics and its suppressor gene.Twelve nude mice were randomly divided into three groups:WiT49 model group,WIT49-miR-20a-5p overexpression group and WIT49-miR-20a-5p knockdown group.The tumor mass and volume of each group were detected by tumor formation experiment in nude mice.real time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-20a-5p,NFKBIB and NF-κB in each group;CCK-8 cell proliferation assay was used to verify the proliferation of tumor cells in each group.Results miR-20a-5p is highly expressed in nephroblastoma and is positively correlated with the expression of NF-κB.miR-20a-5p and NFKBIB have mutual binding sites and binding effects.In the tumor formation experiment of nude mice,the tumor volume and mass of WIT49-miR-20a-5P overexpression group were significantly increased compared with WiT49 model group,and the difference was statistically significant(P<0.05).In the qRT-PCR test,the expressions of miR-20a-5p and NF-κB in the WIT49-miR-20a-5p overexpression group were higher than those in the WiT49 model group,and NFKBIB expression in the WIT49-miR-20a-5p overexpression group was lower than that in the WiT49 model group,with statistical significance(P<0.05).CCK-8 cell proliferation assay showed that the absorbance of WIT49-miR-20a-5p overexpression group at 24 and 48 hours was higher than that of WiT49 model group,and the absorbance of WIT49-miR-20a-5p knockdown group at 24,48 and 72 hours was lower than that of WiT49 model group,and the difference was statistically significant(P<0.05).Conclusion miR-20a-5p may promote the growth of human nephroblastoma cell WiT49 transplanted tumor in nude mice by regulating NFKBIB activation of NF-κB pathway.
RESUMEN
Propolis is a gelatinous substance processed by western worker bees from the resin of plant buds and mixed with the secretions of the maxillary glands and beeswax. Propolis has extensive biological activities and antitumor effects. There have been few reports about the antitumor effect of propolis against human cutaneous squamous cell carcinoma (CSCC) A431 cells and its potential mechanism. CCK-8 assays, label-free proteomics, RT-PCR, and a xenograft tumor model were employed to explore this possibility. The results showed that the inhibition rate of A431 cell proliferation by the ethanol extract of propolis (EEP) was dose-dependent, with an IC50 of 39.17 µg/mL. There were 193 differentially expressed proteins in the EEP group compared with the control group (p < 0.05), of which 103 proteins (53.37%) were upregulated, and 90 proteins (46.63%) were downregulated. The main three activated and suppressed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were extracellular matrix (ECM)-receptor interaction, amoebiasis, cell adhesion molecules (CAMs), nonalcoholic fatty liver disease (NAFLD), retrograde endocannabinoid signaling, and Alzheimer's disease. The tumor volume of the 100 mg/kg EEP group was significantly different from that of the control group (p < 0.05). These results provide a theoretical basis for the potential treatment of human CSCC A431 cell tumors using propolis.